Nucleic acid aptamer specifically combined with cardiac troponin I as well as screening method and application of nucleic acid aptamer

Abstract

The invention discloses a nucleic acid aptamer specifically combined with cardiac troponin I as well as a screening method and application of the nucleic acid aptamer. Belongs to the technical field of molecular biological detection analysis. According to the invention, molecular docking simulation and circular dichroism are used for screening the aptamer which is most stable after being combined with cardiac troponin I and has the largest conformational change. The aptamer Apt-36 obtained by screening is fixed on the surface of a gold electrode after being modified with a disulfide bond and methylene blue, and an electrochemical aptamer sensor is constructed. After the cardiac troponin I is added, the aptamer is induced to generate conformational change, the distance between the redox reporter molecule and the surface of the electrode is changed, and an electrochemical signal easy to detect is generated, so that the cardiac troponin I is analyzed and detected. The sensitivity of the sensor is effectively improved, the detection limit is as low as 1 pg / mL, and the sensor can be directly used for detecting cardiac troponin I in a complex biological sample. The kit has a wide application prospect in clinical diagnosis of acute myocardial infarction.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biological detection and analysis, and in particular relates to a nucleic acid aptamer specifically binding to cardiac troponin I and a screening method and application thereof. Background Art

[0002] Acute myocardial infarction (AMI) has been listed by the World Health Organization as one of the leading causes of death worldwide, and is expected to cause 23 million deaths by 2030. Therefore, the development of a rapid, accurate, and low-cost method for the diagnosis of AMI has aroused great interest among researchers. Currently, some cardiac biomarkers, such as cardiac troponin I (cTnI), myoglobin (Mb), creatine kinase Mb (CK-Mb), and C-reactive protein (C-RP), have been used to diagnose AMI. Compared with other biomarkers, cTnI has higher specificity and sensitivity in the diagnosis of AMI. The reason is that cTnI is released into the human body after the occurrence of AMI and is released into the blood circulation at a very low concentration (critical value 0.01-0.1 ng / mL) after 2-3 hours, reaching a peak within 24 hours. Therefore, accurate and ultrasensitive detection of cTnI provides a valuable method for the diagnosis of early AMI.

[0003] Various sensing methods have been reported for the detection of cTnI, such as surface plasmon resonance (SPR), optical sensing, enzyme-linked immunosorbent assay (ELISA), and electrochemistry, in which the detection elements include antibodies, aptamers, DNA, and RNA. Among these methods, ELISA has high sensitivity and accuracy and has been widely used in clinical cTnI determination. However, this method uses antibodies as recognition elements, which are easily affected by temperature, salt concentration, and pH, have high production costs, and are difficult to modify. In addition, this method has complex operating steps, requires professional operators, is inconvenient, and time-consuming. In recent years, nucleic acid aptamers have become a substitute for antibodies in therapeutic and diagnostic applications due to their high stability, long shelf life, low cost, and easy modification.

[0004] The original chain of the nucleic acid aptamer Apt-40 for cardiac troponin I (cTnI) screened out in the prior art report has 40 bases, its secondary structure free energy is -5.93 kcal / mol, and the structure is relatively stable, and the sequence needs to be redesigned to achieve a larger conformational change. Therefore, the nucleic acid aptamer Apt-40 sequence is re-shortened and designed to destroy the double-stranded stem structure of the nucleic acid aptamer and reduce its stability. In the absence of the target, it is conducive to the formation of the unfolded state of the nucleic acid aptamer, while retaining the ability to recognize and bind to the target, and also increasing the conformational change of the nucleic acid aptamer when binding to cTnI, which is suitable for the preparation of E-AB sensors. Summary of the invention

[0005] The purpose of the present invention is to provide a nucleic acid aptamer that specifically binds to cardiac troponin I and a screening method and application thereof in view of the defects of the prior art. The nucleic acid aptamer has strong specificity and good stability. The electrochemical aptamer sensor prepared has a simple and fast analysis process and can be used for on-site rapid detection of cardiac troponin I (cTnI).

[0006] To achieve the above object, the present invention adopts the following technical solution: A nucleic acid aptamer that specifically binds to cardiac troponin I, wherein the nucleic acid aptamer comprises a characteristic sequence as shown in any one of Apt-38, Apt-36, and Apt-34; The characteristic sequence of Apt-38 is: 5′-GTGCAGTACGCCAACCTT TCTCATGCGCTGCCCCTCTT-3′; the characteristic sequence of Apt-36 is: 5′-TGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCT-3′; the characteristic sequence of Apt-34 is: 5′-GCAGTACGCCAACCTTTCTCATGCGCTGCCCCTC-3′.

[0007] Furthermore, the binding energy of the above-mentioned nucleic acid aptamers to cardiac troponin I satisfies: Apt-34>Apt-38>Apt-36.

[0008] Furthermore, the above-mentioned method for screening nucleic acid aptamers comprises the following steps: Step 1) shortening design, performing two-dimensional structural shortening design on the characteristic sequence of the original chain of Apt-40 to destroy the original double-stranded stem structure and increase its free energy; the characteristic sequence of Apt-40 is 5′-CGT GCA GTA CGC CAA CCT TTC TCA TGC GCT GCC CCT CTT A-3′ Step 2) molecular docking simulation, screening out the shortened characteristic sequence whose binding energy with the cardiac troponin I is less than that of the original chain of the nucleic acid aptamer Apt-40 from step 1) through molecular docking simulation calculation, and the characteristic sequence obtained by screening is any one of Apt-38, Apt-36, and Apt-34; Step 3) The nucleic acid aptamer containing the characteristic sequence obtained in step 2) is combined with cardiac troponin I, and circular dichroism is used to screen out the nucleic acid aptamer with the maximum change value of the positive characteristic peak and the negative characteristic peak near 280 nm and 250 nm before and after the combination.

[0009] The nucleic acid aptamer is used to analyze and detect cardiac troponin I, a biomarker of acute myocardial infarction.

[0010] An electrochemical aptamer sensor comprising the nucleic acid aptamer.

[0011] Furthermore, the structure of the electrochemical aptamer sensor includes nucleic acid aptamer Apt-36.

[0012] Furthermore, the electrochemical aptamer sensor is used in detecting cardiac troponin I.

[0013] Furthermore, the application of the electrochemical aptamer sensor comprises the following steps: (1) Preparation of electrochemical aptamer sensor: The thiolated aptamer Apt-36 is self-assembled on the surface of a nanoporous gold electrode, and the excess sites are occupied by a blocking agent, and then rinsed and stored in a pH 7.4 phosphate buffer solution, wherein the thiolated aptamer Apt-36 is modified with a disulfide bond and a methylene blue group at both ends of the aptamer Apt-36; (2) Basic sample test: the electrochemical aptamer sensor obtained in step (1) is placed in the biological liquid sample solution to be tested together with the reference electrode Ag / AgCl and the counter electrode platinum wire as the working electrode, and the square wave voltammetric current signal I is tested under low frequency and high frequency conditions when there is no cardiac troponin I. blank ; (3) Establishing a concentration-response curve, adding cardiac troponin I to the biological fluid sample solution to be tested in step (2) in batches, and testing the square wave voltammetric current signal I of the biological fluid sample solution to be tested at different concentrations of cardiac troponin I under low frequency and high frequency conditions, respectively. target , Signal gain (S) = (I target – I blank ) / I blank × 100 % (1), defined as signal gain S; or, further defined by kinetic difference KDM signal (D) = S 高频 – S 低频 (2) Calculate the KDM signal at different concentrations, i.e., D; and establish a concentration-response curve of the electrochemical aptamer sensor according to the relationship between the signal gain S or the KDM (D) and the concentration; Determination of calibration parameters: The concentration-response curve obtained in step (3) is fitted using the Hill equation (3) to determine the calibration parameter K of the electrochemical aptamer sensor. 1 / 2 , Y start , Y end , n; the Hill equation is:

[0014] Where Y is S or D Unknown sample concentration test: The calibration parameter K obtained in step (4) is 1 / 2 , Y start , Y end , n is substituted into the Hill equation of this type of electrochemical aptamer sensor again, and after rearrangement (4), the concentration of unknown samples can be quantitatively detected;

[0015] Furthermore, the blocking agent used in the above step (1) can be any one of 6-mercapto-1-hexanol (MCH) and 2-mercaptoethanol (MCE) alcohol thiol derivatives, cysteamine hydrochloride (CHC), sodium diethyldithiocarbamate (DEDTC), and bovine serum albumin (BSA).

[0016] The technical principle of the present invention is: Since the original chain of the nucleic acid aptamer Apt-40 for cardiac troponin I (cTnI) screened out in the prior art report has 40 bases, its secondary structure free energy is -5.93 kcal / mol, and the structure is relatively stable. To address this problem, the nucleic acid aptamer Apt-40 sequence was re-shortened and designed to destroy the double-stranded stem structure of the nucleic acid aptamer and reduce its stability to achieve a larger conformational change; in the absence of the target, it is conducive to the formation of the unfolded state of the nucleic acid aptamer, while retaining the ability to recognize and bind to the target, and also increasing the conformational change of the nucleic acid aptamer when binding to cTnI, which is suitable for the preparation of E-AB sensors. Molecular docking simulation and circular dichroism spectroscopy were used to screen the most stable aptamer with the largest conformational change after binding to cardiac troponin I.

[0017] The advantages of the present invention are: 1. The electrochemical aptamer sensor prepared with the selected aptamer Apt-36 has a high pg / mL sensitivity and a detection limit of 1 pg / mL, which meets the clinical detection requirements of the concentration of cTnI, a biomarker of myocardial infarction (the normal critical value is 0.03-0.13 ng / mL, and >0.5 ng / mL may indicate myocardial infarction).

[0018] 2. In addition, the sensor is easy to operate. Clinicians and even patients themselves can detect cTnI in the blood and get the results quickly within one minute, avoiding long waiting time and delaying the diagnosis and treatment of the patient's condition. Therefore, the sensor is expected to achieve early screening of acute myocardial infarction in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Schematic diagram of docking simulation of different aptamers before and after binding to cTnI. A: Apt-40; B: Apt-38; C: Apt-36; D: Apt-34.

[0020] Figure 2 CD images of different aptamers before and after binding to cTnI at different concentrations. A: Apt-38; B: Apt-36; C: Apt-34.

[0021] Figure 3 The aptamer tailoring design process and aptamer Apt-36 were used to construct the cTnI electrochemical aptasensor.

[0022] Figure 4 Electrochemical aptamer sensor for detecting cTnI in serum. A, B: SWV curves after adding different concentrations of cTnI in serum; C: Concentration-response curve of cTnI sensor in serum; D: Recovery of cTnI sensor in serum.

[0023] Figure 5 Electrochemical aptamer sensor for detecting cTnI in blood. A, B: SWV curves after adding different concentrations of cTnI into the blood; C: Concentration-response curve of cTnI sensor in blood; D: Recovery of cTnI sensor in blood for analysis and detection. DETAILED DESCRIPTION

[0024] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further explained below in combination with specific implementation methods. However, the following examples are merely examples of the present invention and do not represent the scope of protection of the rights limited by the present invention. The scope of protection of the present invention shall be subject to the claims.

[0025] Example 1 The original chain of the nucleic acid aptamer Apt-40 for cardiac troponin I (cTnI) screened out in the prior art report has 40 bases, its secondary structure free energy is -5.93 kcal / mol, and the structure is relatively stable, and the sequence needs to be redesigned to achieve a larger conformational change. Therefore, the nucleic acid aptamer Apt-40 sequence is re-shortened and designed to destroy the double-stranded stem structure of the nucleic acid aptamer and reduce its stability. In the absence of the target, it is conducive to the formation of the unfolded state of the nucleic acid aptamer, while retaining the ability to recognize and bind to the target, and also increasing the conformational change of the nucleic acid aptamer when binding to cTnI, which is suitable for the preparation of E-AB sensors.

[0026] When the original chain of Apt-40 was shortened and designed into Apt-38, Apt-36, and Apt-34 (Table 1); as the aptamer was shortened, the free energy of its secondary structure increased, thereby destroying the structure of Apt-40 and making it prone to conformational changes. However, this may increase the binding energy of the aptamer with the target, leading to thermodynamic instability and reduced affinity; faced with the dilemma of "huge conformational changes" and "thermodynamic instability leading to decreased affinity", we performed molecular docking simulations on the binding of the four aptamers to cTnI ( Figure 1 ). The results showed that the binding energies of aptamers Apt-40, Apt-38, Apt-36, and Apt-34 were -7.3 kcal / mol, -6.9 kcal / mol, -7.5 kcal / mol, and -6.8 kcal / mol, respectively. The selected aptamer Apt-36 had the lowest binding energy with cTnI, which ensured its high affinity, and its high free energy also provided the possibility of large conformational changes.

[0027] Table 1 Predicted secondary structures and free energies of different nucleic acid aptamers

[0028] Example 2 To determine the rationality of the aptamer design, we further investigated whether conformational changes occurred after different aptamers bound to cTnI. The circular dichroism (CD) spectra of different aptamers in the presence or absence of cTnI were tested. The unmodified aptamer was first diluted to 6 μM, heated in an incubator at 95 °C for 5 min, and then quickly placed at room temperature for rapid cooling. Finally, different concentrations of cTnI were added to make the final cTnI concentrations of 0.5 ng / mL, 1 ng / mL, and 5 ng / mL, respectively, and the solution was used for CD testing (parameters were 5 mdeg sensitivity, 50 nm / min scan speed, 2 nm bandwidth, and 1 nm data spacing). Figure 2 The CD experimental results in the results show that the nucleic acid aptamer Apt-36 has positive and negative characteristic peaks near 280 nm and 250 nm, respectively, indicating that the nucleic acid aptamer is a typical B-type DNA. It is well known that the DNA base stacking forms a positive cotton effect peak, and its helical structure forms a negative cotton effect peak. In the presence of target cTnI, the positive and negative characteristic cotton effect peaks at wavelengths of 280 nm and 250 nm changed significantly, indicating that the binding of nucleic acid aptamer Apt-36 to cTnI caused significant conformational changes, which provided the possibility for the E-AB sensor to detect cTnI. Compared with nucleic acid aptamer Apt-36, the positive and negative characteristic peaks of Apt-38 and Apt-34 after binding to cTnI changed less at 280 nm and 250 nm.

[0029] Example 3 Construction of electrochemical aptamer sensor The best aptamer Apt-36 was modified with disulfide bonds and methylene blue (MB) at both ends (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to construct an electrochemical aptamer sensor for detecting cTnI in different biological samples (serum and blood). The modified truncated aptamer was fixed on the surface of the gold electrode to construct an electrochemical aptamer (E-AB) sensor. After the addition of cTnI, the aptamer underwent a significant conformational transition, the distance between the redox reporter molecule methylene blue and the electrode surface became smaller, the square wave voltammetry (SWV) signal was enhanced, and the SWV signal decreased with the increase of cTnI concentration, so that the relationship curve between the signal gain (S) and the cTnI concentration could be obtained.

[0030] Calculate the signal gain (S) using the following equation:

[0031] Among them I target and I blank Square wave voltammetric peak currents in the presence and absence of cTnI at frequencies of 5–500 Hz, respectively.

[0032] Kinetic difference measurement (KDM) was used to correct for baseline drift in complex biological samples.

[0033]

[0034] S 高频 and S 低频 They represent the signal gains obtained at high frequencies (50-500 Hz) and low frequencies (5-50 Hz), respectively.

[0035] Establishing a concentration-response curve of the electrochemical aptamer sensor according to the relationship between the signal gain (S) or KDM signal (D) and the concentration obtained above; Next, the obtained concentration-signal response curve was fitted by the Hill equation (Equation 3) to obtain the relevant parameters of the sensor. The Hill equation is as follows:

[0036] Where Y is S or D Where n represents the Hill coefficient; D start and D end Respectively represent the minimum and maximum values ​​of the KDM signal. d is the midpoint of the concentration-response curve. Substituting the KDM signal into the rearranged form of equation (3) (Equation 4) the target concentration can be calculated.

[0037] Sensor preparation process: First, nanoporous gold electrodes were prepared by electrochemical alloying / dealloying technology. 2 The benzyl alcohol solution (1.5 M) was heated to 125 °C, and a three-electrode system (reference electrode: Ag / AgCl, counter electrode: platinum wire) was placed in it, and 50 CV scans were performed (scanning range was -0.72 - 1.88 V, and the scanning rate was 0.01 V / s). The gold electrode was then ultrasonically cleaned in acetone, ethanol, and deionized water in sequence, and the electrode was immersed in 0.5 M HCl solution for dealloying. Finally, the nanoporous gold electrode was electrochemically cleaned (0.5 M NaOH, CV scan, potential -0.35 V to -1.35 V, scanning rates were 4 V / s and 2 V / s, respectively, and the number of cycles was 200 and 1000, respectively) and activated (0.5 MH 2 SO 4 , CV scan, potential 0.2 V to 1.6 V, scan rates 2 V / s and 0.1 V / s, cycle 20 times) for subsequent use.

[0038] The selected aptamer Apt-36 (1 μL, 100 μM) was incubated with tri(2-carboxyethyl)phosphine (9 μL, 10 mM) (TCEP) at room temperature in the dark for 1 hour to reduce the disulfide bonds modified by the aptamer, and then diluted to 50 nM with phosphate buffer (pH 7.4). The gold electrode, which was electrochemically cleaned and activated in sodium hydroxide and sulfuric acid, was placed in a 50 nM aptamer solution and incubated at room temperature in the dark for 1 hour to fix the aptamer on the surface of the nanoporous gold electrode through the Au-S bond self-assembled monolayer; then the blocking agent mercaptohexanol (3 mM MCH) was placed in the room temperature in the dark for one hour to occupy the excess sites on the electrode surface. Finally, it was rinsed with deionized water and the electrode was stored in a phosphate buffer solution (pH 7.4) to prepare an electrochemical aptamer (E-AB) sensor.

[0039] Application Example 1 Detection of cTnI in serum by electrochemical aptamer sensor The electrochemical aptamer (E-AB) sensor prepared in Example 3 was used to detect cTnI in serum.

[0040] First, square wave voltammetry (SWV) scanning was performed at frequencies of 250 Hz and 20 Hz (scan range 0 - -0.45 V), and the SWV response signal of serum without cTnI (purchased from Merck) was obtained as blank I. blankThen, different concentrations of cTnI (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 and 100 ng / mL) were added to the serum to obtain I target The signal gain (S) at different concentrations was calculated using Formula 1, and the concentration-response curves at frequencies of 250 Hz and 20 Hz were obtained by fitting the Hill equation (Formula 3). Figure 4 C), and the parameter K of the curve can be determined d , S start ,S end , n (respectively: 0.34 ng / mL, 6.08 %, 49.04 %, 0.55).

[0041] In order to further increase the E-AB sensor signal gain and reduce K d , and further calculate KDM (D) at different concentrations by kinetic difference. The concentration-response curve is obtained by fitting it with the Hill equation (Formula 3), and the parameter K of the calibration curve is determined at the same time. d , D start , D end , n (respectively: 0.13 ng / mL, 9.13 %, 73.59 %, 0.26).

[0042] Since these parameters are universal in this type of sensor, quantitative detection can be performed directly on the serum sample to be tested. For example, the sensor is used to detect a serum sample with an unknown cTnI concentration. The electrode is placed in the serum sample to be tested and the KDM (D) is 50%. d , D start , D end , n (respectively: 0.13 ng / mL, 9.13%, 73.59%, 0.26), by substituting into equation (4), cTnI and [C Target ] has a concentration of 1.07 ng / mL.

[0043] Figure 4 The detection range of cTnI in serum was 0.001 to 100 ng / mL, the detection limit was 0.001 ng / mL, and the recovery rate was 75.3~105.6%. Figure 4 (A, B) SWV curves after adding different concentrations of cTnI in serum. (C) Concentration-response curve of cTnI sensor in serum. (D) Recovery of cTnI sensor in serum.

[0044] Application Example 2 Electrochemical aptamer sensor for detecting cTnI in blood The electrochemical aptamer (E-AB) sensor prepared in Example 3 was used to detect cTnI in blood.

[0045] First, square wave voltammetry (SWV) scanning (scanning range 0 - -0.45 V) was performed at frequencies of 250 Hz and 20 Hz to obtain the SWV response signal of blood without cTnI (purchased from Zhengzhou Pingrui Biology) as blank I blank Then, different concentrations of cTnI (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 and 100 ng / mL) were added to the blood to obtain I target The gain (S) at different concentrations was calculated using Formula 1, and the concentration-response curves ( Figure 5 C), the experimental parameter K of the calibration curve was determined d , S start , S end , n (respectively: 5.83 ng / mL, 4.80 %, 50.36 %, 0.34).

[0046] In addition, in order to further increase the E-AB sensor signal gain and reduce K d We further calculated KDM (D) at different concentrations by kinetic difference. The concentration-response curve was obtained by fitting it with the Hill equation (Formula 3), and the parameter K of the calibration curve was determined at the same time. d , D start , D end , n (respectively: 1.51 ng / mL, 10.03%, 106.41%, 0.33).

[0047] Since these parameters are universal in this type of sensor, quantitative detection can be performed directly on the blood sample to be tested. For example, the sensor is used to detect a blood sample with an unknown cTnI concentration. The electrode is placed in the blood sample to be tested and the KDM (D) is 80%. d , D start , D end , n (respectively: 1.51 ng / mL, 10.03%, 106.41%, 0.33), by substituting into equation (4), cTnI and [C Target ] has a concentration of 28.94 ng / mL.

[0048] Figure 5For cTnI testing in blood, the detection range is 0.001 to 100 ng / mL, the detection limit is 0.001 ng / mL, and the recovery rate is 72.5~125.8%.

[0049] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A nucleic acid aptamer that specifically binds to cardiac troponin I, characterized in that: The nucleic acid aptamer includes a characteristic sequence as shown in any one of Apt-38, Apt-36, and Apt-34; The characteristic sequence of Apt-38 is: 5′-GTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTT-3′; the characteristic sequence of Apt-36 is: 5′-TGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCT-3′; the characteristic sequence of Apt-34 is: 5′-GCAGTACGCCAACCTTTCTCATGCGCTGCCCCTC-3′.

2. The nucleic acid aptamer according to claim 1, characterized in that: The binding energy of the nucleic acid aptamer to cardiac troponin I satisfies the following conditions: Apt-34 > Apt-38 > Apt-36.

3. A method for screening nucleic acid aptamers according to claim 1, characterized in that: The following steps are involved: Step 1) Shortening design: Perform two-dimensional shortening design on the characteristic sequence of the original chain of Apt-40 to destroy the original double-stranded stem structure and increase its free energy; Step 2) Molecular docking simulation: Through molecular docking simulation calculation, a shortened characteristic sequence having a binding energy with cardiac troponin I smaller than the original chain of the nucleic acid aptamer Apt-40 is screened out from step 1), and the screened characteristic sequence is any one of Apt-38, Apt-36, and Apt-34; Step 3) The nucleic acid aptamer containing the characteristic sequence screened in step 2) is combined with cardiac troponin I, and circular dichroism is used to screen out the nucleic acid aptamer with the maximum change value of the positive characteristic peak and the negative characteristic peak near 280 nm and 250 nm before and after the binding.

4. The use of the nucleic acid aptamer according to claim 1, characterized in that: The nucleic acid aptamer is used for analyzing and detecting cardiac troponin I, a biomarker of acute myocardial infarction. 5 . An electrochemical aptamer sensor comprising the nucleic acid aptamer according to claim 1 .

6. The electrochemical aptamer sensor according to claim 5, characterized in that: The structure of the electrochemical aptamer sensor includes nucleic acid aptamer Apt-36.

7. Use of the electrochemical aptamer sensor as claimed in claim 5 in detecting cardiac troponin I.

8. The use of the electrochemical aptamer sensor as claimed in claim 7, characterized in that: The following steps are involved: (1) Preparation of electrochemical aptamer sensor: The thiolated aptamer Apt-36 is self-assembled on the surface of a nanoporous gold electrode, and the excess sites are occupied by a blocking agent, and then rinsed and stored in a pH 7.4 phosphate buffer solution, wherein the thiolated aptamer Apt-36 is modified with a disulfide bond and a methylene blue group at both ends of the aptamer Apt-36; (2) Basic sample test: the electrochemical aptamer sensor obtained in step (1) is placed in the biological liquid sample solution to be tested together with the reference electrode Ag / AgCl and the counter electrode platinum wire as the working electrode, and the square wave voltammetric current signal I is tested under low frequency and high frequency conditions when there is no cardiac troponin I. blank ; (3) Establishing a concentration-response curve, adding cardiac troponin I to the biological fluid sample solution to be tested in step (2) in batches, and testing the square wave voltammetric current signal I of the biological fluid sample solution to be tested at different concentrations of cardiac troponin I under low frequency and high frequency conditions, respectively. target , Signal gain (S) = (I target – I blank ) / I blank × 100 % (1), defined as the signal gain S; or, further defined by kinetic difference KDM signal (D) = S 高频 –S 低频 (2) Calculate the KDM signal at different concentrations, i.e., D; and establish a concentration-response curve of the electrochemical aptamer sensor based on the relationship between the signal gain S or the KDM signal and the concentration; Determination of calibration parameters: The concentration-response curve obtained in step (3) is fitted using the Hill equation (3) to determine the calibration parameter K of the electrochemical aptamer sensor. 1 / 2 , Y start , Y end , n; the Hill equation is: Where Y is S or D Unknown sample concentration test: The calibration parameter K obtained in step (4) is 1 / 2 , Y start , Y end , n is substituted into the Hill equation of this type of electrochemical aptamer sensor again, and after rearrangement (4), the concentration of unknown samples can be quantitatively detected; 。 9. The use of the electrochemical aptamer sensor according to claim 8, characterized in that: The blocking agent used in step (1) is any one of 6-mercapto-1-hexanol and 2-mercaptoethanol alcohol thiol derivatives, cysteamine hydrochloride, sodium diethyldithiocarbamate, and bovine serum albumin.

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